Project Summary The goal of this application is to develop a single vesicle technology (SVT) for exosome surface protein profiling based on surface enhanced Raman scattering (SERS) imaging in conjunction with direct molecular exosome capture from diluted biofluids. Single exosome profiling of surface proteins would provide unprecedented insight into biological events and invaluable information for biomarker discovery. It can probe tumor-derived exosomes in the presence of abundant non-tumor exosomes, providing sensitive, precise, and quantitative information superior to bulk methods. However, single exosome protein profiling is challenging due to the small size and low abundance of antigens on individual exosomes as well as the difficulties in isolation of pure exosomes for downstream analysis. To address the technical challenges, we use highly sensitive and specific SERS nanotags to label and image exosomal surface proteins. We have developed a surface chemistry that can molecularly capture exosomes directly from diluted biofluids based on exosomal CD81 marker expression. Our method only requires microliter of extremely diluted plasma (typically 100-fold dilution), which is over 150 times less than the bulk enzyme-linked immunosorbent assay. Due to the advantages in simplicity, sensitivity, efficiency, and sample consumption, the SERS-SVT, if successful, would substantially improve the analytical performance for molecular characterization of exosomes for cancer research and accelerate the progress in the exosome field in terms of biomarker discovery and clinical translation. Our SERS-SVT will be developed and tested for breast cancer diagnostics through the following three aims: 1) Develop and optimize SERS-SVT methodologies for single exosome protein profiling; 2) Characterize and validate SERS-SVT with cell-derived exosomes in the breast cancer model; and 3) Apply SERS-SVT to examine the potential of exosomes for cancer diagnostics at different stages using HER2- positive breast cancer as the disease model. We have extensively developed the SERS-SVT instrumentation and methodologies including specific and stable SERS gold nanorod (AuNR) tags for exosomal protein labeling and a single exosome dual imaging analysis (SEDIA) software for fast image analysis. We further showed that SERS-SVT profiling, but not ELISA, of HER2 expression on CD81- positive exosomes detected early-stage HER2-positive breast cancer. Exosomal CD24 can differentiate locally advanced stage from early-stage. The proposed studies will investigate HER2, CD24, and some other markers involving in different aspects of cancer development and metastasis to examine whether the use of composite biomarkers can detect HER2-positive BC and inform the extent of cancer at diagnostics through plasma exosomes. Five undergraduates will work on this project. Undergraduates will be trained in various aspects of nanomedicine from nanomaterials synthesis to biomarker detection.